Alfredo Darío Espinosa Brito: insigne de las ciencias médicas

Introducción: Cuando se habla del símbolo de las generaciones de especialistas que se dedican a la Medicina Interna es necesario referirse al Doctor Alfredo Darío Espinosa Brito, destacado internista cienfueguero que cuenta con una fecunda trayectoria merecedora del reconocimiento de la comunidad médica. Objetivo: Describir los acontecimientos significativos de su vida y obra. Métodos: Se realizó una investigación de tipo descriptiva, que utiliza la historia de vida para lograr su propósito. Fue empleado el método teórico histórico-lógico; para la recolección de datos, se realizó una revisión documental y el testimonio directo de la voz del profesor como fuente primaria de obtención de la información. Desarrollo: los progenitores le inculcaron nobles principios de honradez que definieron su personalidad. Tuvo una trayectoria estudiantil sobresaliente marcada por ideas progresistas. Cuenta con una brillante carrera profesional merecedora de múltiples condecoraciones, pues su influencia ha sido esencial en la enseñanza del método clínico. Conclusiones: El doctor Espinosa ha contribuido sobremanera al desarrollo de la Medicina Interna, la Terapia Intensiva y la Geriatría en el territorio, además de realizar importantes aportes docentes, investigativos, teóricos y prácticos al avance de la enseñanza y de la Salud Pública en Cuba.

Introduction: When talking about the symbol of the generations of specialists dedicated to Internal Medicine, it is necessary to refer to Dr. Alfredo Darío Espinosa Brito, an outstanding internist from Cienfuegos who has a fruitful career deserving the recognition of the medical community. Objective: To describe the significant events of his life and work. Methods: A descriptive research was carried out, using life history to achieve its purpose. The historical-logical theoretical method was used; for data collection, a document review and the direct testimony of the professor's voice were used as the primary source of information. Development: His parents instilled in him noble principles of honesty that defined his personality. He had an outstanding student career marked by progressive ideas. He has a brilliant professional career deserving of multiple decorations, as his influence has been essential in the teaching of the clinical method. Conclusions: Dr. Espinosa has contributed greatly to the development of Internal Medicine, Intensive Care and Geriatrics in the territory, in addition to making important teaching, research, theoretical and practical contributions to the advancement of teaching and Public Health in Cuba.

ICAP-1 loss impairs CD8+ thymocyte development and leads to reduced marginal zone B cells in mice.

ICAP-1 regulates ß1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4ß1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8+ cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1-/- spleen T and B cells displayed upregulation of α4ß1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+ - and CD19+ -selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B-cell numbers.

Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers.

Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.

Paradigmatic De Novo GRIN1 Variants Recapitulate Pathophysiological Mechanisms Underlying GRIN1-Related Disorder Clinical Spectrum.

BACKGROUND: GRIN-related disorders (GRD), the so-called grinpathies, is a group of rare encephalopathies caused by mutations affecting GRIN genes (mostly GRIN1, GRIN2A and GRIN2B genes), which encode for the GluN subunit of the N-methyl D-aspartate (NMDA) type ionotropic glutamate receptors. A growing number of functional studies indicate that GRIN-encoded GluN1 subunit disturbances can be dichotomically classified into gain- and loss-of-function, although intermediate complex scenarios are often present. METHODS: In this study, we aimed to delineate the structural and functional alterations of GRIN1 disease-associated variants, and their correlations with clinical symptoms in a Spanish cohort of 15 paediatric encephalopathy patients harbouring these variants. RESULTS: Patients harbouring GRIN1 disease-associated variants have been clinically deeply-phenotyped. Further, using computational and in vitro approaches, we identified different critical checkpoints affecting GluN1 biogenesis (protein stability, subunit assembly and surface trafficking) and/or NMDAR biophysical properties, and their association with GRD clinical symptoms. CONCLUSIONS: Our findings show a strong correlation between GRIN1 variants-associated structural and functional outcomes. This structural-functional stratification provides relevant insights of genotype-phenotype association, contributing to future precision medicine of GRIN1-related encephalopathies.

José María Bermúdez López: símbolo de la medicina interna en Cienfuegos / José María Bermúdez López: An internal medicine symbol in Cienfuegos

RESUMEN La vida de José María Bermúdez López, destacado médico cienfueguero, está llena de pasajes extraordinarios que le confieren el reconocimiento de la comunidad científica internacional. Su mérito trasciende más allá de la profesión, pues no estuvo al margen de las luchas estudiantiles contra Batista durante su etapa universitaria, y mantuvo el compromiso con la Revolución brindando servicios sanitarios en zonas rurales. Sin embargo, existe una escasa divulgación de su trayectoria, incluso en la provincia de Cienfuegos; es por ello que el objetivo de este trabajo es describir los acontecimientos significativos de la vida y obra de este doctor, símbolo de la Medicina Interna. Se realizó una investigación de tipo descriptiva, que utiliza la historia de vida para lograr su propósito. Fueron empleados los métodos teóricos: analítico-sintético, deductivo-inductivo e histórico-lógico; para la recolección de datos se utilizaron la iconografía, la revisión documental y el testimonio directo de la voz del profesor como fuente primaria de obtención de la información. Las bibliografías consultadas fueron escasas, por lo que fue necesario recurrir a las entrevistas orales a profundidad. Se concluye que el doctor José María, durante su trabajo ininterrumpido, ha contribuido de forma exhaustiva al desarrollo de esta especialidad en el territorio mediante un compromiso con las ciencias médicas y con los enfermos. Conocido como "El Profe", con 88 años de edad, aún camina por los pasillos del hospital que fundó, admirado por todos.

ABSTRACT The life of José María Bermúdez López, a prominent Cienfuegos doctor, is full of extraordinary stories that confer him the recognition of the international scientific community. His merit transcends beyond his profession, since he was not on the sidelines of the struggles against Batista during his university years, and he maintained his commitment to the Revolution by providing health services in rural areas. However, there is little disclosure of his career, even in the province of Cienfuegos; That is why the objective of this work is to describe the significant events in the life and work of this doctor, a symbol of Internal Medicine. A descriptive research was carried out, which uses life history to achieve its purpose. Theoretical methods were used: analytical-synthetic, deductive-inductive and historical-logical; For data collection, iconography, documentary review and direct testimony of the teacher's voice were used as the primary source for obtaining information. The bibliographies consulted were scarce, so it was necessary to resort to in-depth oral interviews. It is concluded that Dr. José María, during his continuous work, has contributed exhaustively to the development of this specialty in the territory through a commitment to medical sciences and patients. Known as "El Profe", at 88 years old, he still walks the corridors of the hospital he founded, admired by all.

CD3G or CD3D Knockdown in Mature, but Not Immature, T Lymphocytes Similarly Cripples the Human TCRαß Complex.

The human αß T-cell receptor (TCR) is composed of a variable heterodimer (TCRαß) and three invariant dimers (CD3γε, CD3δε, and ζζ/CD2472). The role of each invariant chain in the stepwise interactions among TCR chains along the assembly is still not fully understood. Despite the high sequence homology between CD3γ and CD3δ, the clinical consequences of the corresponding immunodeficiencies (ID) in humans are very different (mild and severe, respectively), and mouse models do not recapitulate findings in human ID. To try to understand such disparities, we stably knocked down (KD) CD3D or CD3G expression in the human Jurkat T-cell line and analyzed comparatively their impact on TCRαß assembly, transport, and surface expression. The results indicated that TCR ensembles were less stable and CD3ε levels were lower when CD3γ, rather than CD3δ, was scarce. However, both defective TCR ensembles were strongly retained in the ER, lacked ζζ/CD2472, and barely reached the T-cell surface (<11% of normal controls) in any of the CD3 KD cells. This is in sharp contrast to human CD3γ ID, whose mature T cells express higher levels of surface TCR (>30% vs. normal controls). CD3 KD of human T-cell progenitors followed by mouse fetal thymus organ cultures showed high plasticity in emerging immature polyclonal T lymphocytes that allowed for the expression of significant TCR levels which may then signal for survival in CD3γ, but not in CD3δ deficiency, and explain the immunological and clinical disparities of such ID cases.

The recycling of surgical face masks as sound porous absorbers: Preliminary evaluation.

This paper presents the results of an experimental study on the acoustic efficiency of plastic surgical face masks. Since the very high number of disposable masks being used globally on a daily basis to face the Covid19 pandemic is posing new environmental risks, mainly connected to improper disposal, any possible improvements in the management of this waste stream is very important. In this work their potential use as sound porous absorber is discussed. Surgical face masks are mainly made of polypropylene fibers which show good acoustical properties. Their porous structure was studied through the measurement of some non-acoustic properties: bulk density, fiber diameter, porosity, flow resistivity and tortuosity. Moreover, the sound absorption performance of samples, made of scrapped face masks, with different thicknesses was evaluated using an impedance tube according to ISO 10534-2. The results obtained from the sound absorption spectra and two single indexes, Noise Reduction Coefficient and Sound Absorption Average showed a high sound absorption value over a frequency range of interest. Finally, the sound absorption spectra obtained for surgical face masks were compared with those obtained for fibrous materials currently used in building sector, suggesting that this fibrous waste could act as a possible substitute to traditional ones.

Cyclooxygenase 2 Effector Genes as Potential Inflammation-Related Biomarkers for Colorectal Cancer Circulating Tumor Cells Detection by Liquid Biopsy.

Cyclooxygenase 2 (COX2) has been implicated in cancer development and metastasis. We have identified several COX2-regulated inflammation-related genes in human colorectal cancer cells and shown that some of them play important roles in tumor progression. In this work, we have studied the COX2-regulated genes in the mouse colorectal cancer cell line CT26, to find that many are also regulated by COX2 over-expression. On the other hand, we generated a CT26 cell line expressing Gfp and Luciferase, to study tumor growth and metastasis in immunocompetent Balb/c mice. We then collected solid tissue, and blood samples, from healthy and tumor-bearing mice. Using the Parsortix® cell separation system and taking advantage of the fact that the tumor cells expressed Gfp, we were able to identify circulating tumor cells (CTCs) in some of the mice. We compared the mRNA expression levels of Ptgs2 and effector genes in the samples obtained from tumor-bearing or healthy mice, namely, tumor or healthy colon, Ficoll purified buffy coat, and Parsortix-isolated cells to find different patterns between healthy, tumor-bearing mice with or without CTCs. Although for genes like Il15 we did not observe any difference between healthy and tumor-bearing mice in Ficoll or Parsortix samples; others, such as Egr1, Zc3h12a, Klf4, or Nfat5, allowed distinguishing for cancer or CTC presence. Gene expression analysis in Ficoll or Parsortix processed samples, after liquid biopsy, may offer valuable diagnostic and prognostic information and thus should be further studied.

Human T-ALL Xenografts.

Intense chemotherapy regimens of patients diagnosed with T cell acute lymphoblastic leukemia (T-ALL) have proved successful for improving patient's overall survival, especially in children. But still T-ALL treatment remains challenging, since side effects of chemotherapeutic drugs often worsen patient's quality of life, and relapse rates remain significant. Hence, the availability of experimental animal models capable of recapitulating the biology of human T-ALL is obligatory as a critical tool to explore novel promising therapies directed against specific targets that have been previously validated in in vitro assays. For this purpose, patient-derived xenografts (PDX) of primary human T-ALL are currently of great interest as preclinical models for novel therapeutic strategies toward transition into clinical trials. In this chapter, we describe the lab workflow to perform PDX assays, from the initial processing of patient T-ALL samples, genetic in vitro modifications of leukemic cells by lentiviral transduction, inoculation routes, monitoring for disease development, and mouse organ examination, to administration of several treatments.

Downregulation of Glutamine Synthetase, not glutaminolysis, is responsible for glutamine addiction in Notch1-driven acute lymphoblastic leukemia.

The cellular receptor Notch1 is a central regulator of T-cell development, and as a consequence, Notch1 pathway appears upregulated in > 65% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T-ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti-Notch therapies in T-ALL models. In this work, we report that Notch1 upregulation in T-ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1-driven T-ALL both in vitro and in vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1-induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1-driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1-driven leukemia.

Evaluation of a multidisciplinary global health online course in Mexico.

BACKGROUND: Global Health Education (GHE) focuses on training proactive global citizens to tackle health challenges in an increasingly interconnected and interdependent world. Studies show that health professionals in training have reported that GHE has improved their teamwork, responsiveness to contextual factors that impact health, and understanding of health systems; however, there is little research on the impact of GHE courses in undergraduate settings, especially in low and middle-income countries (LMICs). METHODS: Our study analyzes a multidisciplinary online global health course at Tecnologico de Monterrey, México. We conducted a cross-sectional study with pre- and post-design. Students who took the multidisciplinary course of Global Health for Leaders in the Fall of 2019 (n = 153) and Spring of 2020 (n = 348) were selected for this study. Using a five-point Likert scale (strongly agree to strongly disagree), the survey assessed seven competencies as well as questions about course expectations, takeaways, and recommendations to improve the course. We performed descriptive statistical analyses comparing the combined pre-tests (from Fall and Spring cohorts) to the combined post-tests. Fisher's exact test was used to compare the samples. RESULTS: Of the 501 pre-course surveys administered, 456 responses were completed in the pre-course and 435 in the post-course (91% overall response rate). Only 8.7% of the respondents in the pre-course survey strongly agreed that they could describe fundamental aspects of global health such as the Millennium Development Goals or Sustainable Development Goals, in contrast to a 56% of the students who strongly agreed in the post-course survey (p < 0.001). Similar differences were captured in understanding the global burden of disease, social determinants of health, the effects of globalization in health, health systems' goals and functions, and human rights. 38% felt that the course helped them develop a more empathetic perception of the suffering of others experiencing global health-related issues. CONCLUSION: In this study, we have presented our experience in teaching an online global health course for multidisciplinary undergraduates in a LMIC. The competencies reported by our students indicate that the course prepared them to confront complex global health issues.

Predictors of suboptimal adherence to isoniazid preventive therapy among adolescents and children living with HIV.

This study identified factors associated with adherence to a 6-month isoniazid preventive therapy (IPT) course among adolescents and children living with HIV. Forty adolescents living with HIV and 48 primary caregivers of children living with HIV completed a Likert-based survey to measure respondent opinions regarding access to care, quality of care, preferred regimens, perceived stigma, and confidence in self-efficacy. Sociodemographic data were collected and adherence measured as the average of pill counts obtained while on IPT. The rates of suboptimal adherence (< 95% adherent) were 22.5% among adolescents and 37.5% among the children of primary caregivers. Univariate logistic regression was used to model the change in the odds of suboptimal adherence. Independent factors associated with suboptimal adherence among adolescents included age, education level, the cost of coming to clinic, stigma from community members, and two variables relating to self-efficacy. Among primary caregivers, child age, concerns about stigma, and location preference for meeting a community-health worker were associated with suboptimal adherence. To determine whether these combined factors contributed different information to the prediction of suboptimal adherence, a risk score containing these predictors was constructed for each group. The risk score had an AUC of 0.87 (95% CI: 0.76, 0.99) among adolescents and an AUC of 0.76 (95% CI: 0.62, 0.90), among primary caregivers suggesting that these variables may have complementary predictive utility. The heterogeneous scope and associations of these variables in different populations suggests that interventions aiming to increase optimal adherence will need to be tailored to specific populations and multifaceted in nature. Ideally interventions should address both long-established barriers to adherence such as cost of transportation to attend clinic and more nuanced psychosocial barriers such as perceived community stigma and confidence in self-efficacy.

CCR7 as a novel therapeutic target in t-cell PROLYMPHOCYTIC leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a poor prognostic disease with very limited options of efficient therapies. Most patients are refractory to chemotherapies and despite high response rates after alemtuzumab, virtually all patients relapse. Therefore, there is an unmet medical need for novel therapies in T-PLL. As the chemokine receptor CCR7 is a molecule expressed in a wide range of malignancies and relevant in many tumor processes, the present study addressed the biologic role of this receptor in T-PLL. Furthermore, we elucidated the mechanisms of action mediated by an anti-CCR7 monoclonal antibody (mAb) and evaluated whether its anti-tumor activity would warrant development towards clinical applications in T-PLL. Our results demonstrate that CCR7 is a prognostic biomarker for overall survival in T-PLL patients and a functional receptor involved in the migration, invasion, and survival of leukemic cells. Targeting CCR7 with a mAb inhibited ligand-mediated signaling pathways and induced tumor cell killing in primary samples. In addition, directing antibodies against CCR7 was highly effective in T-cell leukemia xenograft models. Together, these findings make CCR7 an attractive molecule for novel mAb-based therapeutic applications in T-PLL, a disease where recent drug screen efforts and studies addressing new compounds have focused on chemotherapy or small molecules. SUPPLEMENTARY INFORMATION: Supplementary information accompanies this paper at 10.1186/s40364-020-00234-z.

Direct Determination of Pyrazinamide (PZA) Susceptibility by Sputum Microscopic Observation Drug Susceptibility (MODS) Culture at Neutral pH: the MODS-PZA Assay.

Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 µg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 µg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 µg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.

Evaluation of therapeutic targeting of CCR7 in acute graft-versus-host disease.

Graft-versus-host disease (GVHD) is the main complication after allogeneic hematopoietic stem cell transplantation. We previously unveiled a correlation between proportions of C-C motif chemokine receptor 7 (CCR7)+ T cells in the apheresis and the risk of developing GVHD. We wanted to evaluate in vivo whether apheresis with low proportion of CCR7+ cells or treatment with an anti-human CCR7 monoclonal antibody (mAb) were suitable strategies to prevent or treat acute GVHD in preclinical xenogeneic models. Therapeutic anti-CCR7 mAb was the most effective strategy in both prophylactic and therapeutic settings where antibody drastically reduced in vivo lymphoid organ infiltration of donor CCR7+ T cells, extended lifespan and solved clinical signs. The antibody neutralized in vitro migration of naïve and central memory T cells toward CCR7 ligands and depleted target CCR7+ subsets through complement activation. Both mechanisms of action spared CCR7- subsets, including effector memory and effector memory CD45RA+ T cells which may mediate graft versus leukemia effect and immunity against infections. Accordingly, the numbers of donor CCR7+ T cells in the apheresis were not associated to cytomegalovirus reactivation or the recurrence of the underlying disease. These findings provide a promising new strategy to prevent and treat acute GVHD, a condition where new specific, safety and effective treatment is needed.

Hippocampus segmentation on noncontrast CT using deep learning.

PURPOSE: Accurate segmentation of the hippocampus for hippocampal avoidance whole-brain radiotherapy currently requires high-resolution magnetic resonance imaging (MRI) in addition to neuroanatomic expertise for manual segmentation. Removing the need for MR images to identify the hippocampus would reduce planning complexity, the need for a treatment planning MR imaging session, potential uncertainties associated with MRI-computed tomography (CT) image registration, and cost. Three-dimensional (3D) deep convolutional network models have the potential to automate hippocampal segmentation. In this study, we investigate the accuracy and reliability of hippocampal segmentation by automated deep learning models from CT alone and compare the accuracy to experts using MRI fusion. METHODS: Retrospectively, 390 Gamma Knife patients with high-resolution CT and MR images were collected. Following the RTOG 0933 guidelines, images were rigidly fused, and a neuroanatomic expert contoured the hippocampus on the MR, then transferred the contours to CT. Using a calculated cranial centroid, the image volumes were cropped to 200 × 200 × 35 voxels, which were used to train four models, including our proposed Attention-Gated 3D ResNet (AG-3D ResNet). These models were then compared with results from a nested tenfold validation. From the predicted test set volumes, we calculated the 100% Hausdorff distance (HD). Acceptability was assessed using the RTOG 0933 protocol criteria, and contours were considered passing with HD ≤ 7 mm. RESULTS: The bilateral hippocampus passing rate across all 90 models trained in the nested cross-fold validation was 80.2% for AG-3D ResNet, which performs with a comparable pass rate (P = 0.3345) to physicians during centralized review for the RTOG 0933 Phase II clinical trial. CONCLUSIONS: Our proposed AG-3D ResNet's segmentation of the hippocampus from noncontrast CT images alone are comparable to those obtained by participating physicians from the RTOG 0933 Phase II clinical trial.

Specific NOTCH1 antibody targets DLL4-induced proliferation, migration, and angiogenesis in NOTCH1-mutated CLL cells.

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.

Notch1 signaling in NOTCH1-mutated mantle cell lymphoma depends on Delta-Like ligand 4 and is a potential target for specific antibody therapy.

BACKGROUND: NOTCH1 gene mutations in mantle cell lymphoma (MCL) have been described in about 5-10% of cases and are associated with significantly shorter survival rates. The present study aimed to investigate the biological impact of this mutation in MCL and its potential as a therapeutic target. METHODS: Activation of Notch1 signaling upon ligand-stimulation and inhibitory effects of the monoclonal anti-Notch1 antibody OMP-52M51 in NOTCH1-mutated and -unmutated MCL cells were assessed by Western Blot and gene expression profiling. Effects of OMP-52M51 treatment on tumor cell migration and tumor angiogenesis were evaluated with chemotaxis and HUVEC tube formation assays. The expression of Delta-like ligand 4 (DLL4) in MCL lymph nodes was analyzed by immunofluorescence staining and confocal microscopy. A MCL mouse model was used to assess the activity of OMP-52M51 in vivo. RESULTS: Notch1 expression can be effectively stimulated in NOTCH1-mutated Mino cells by DLL4, whereas in the NOTCH1-unmutated cell line JeKo-1, less effect was observed upon any ligand-stimulation. DLL4 was expressed by histiocytes in both, NOTCH1-mutated and -unmutated MCL lymph nodes. Treatment of NOTCH1-mutated MCL cells with the monoclonal anti-Notch1 antibody OMP-52M51 effectively prevented DLL4-dependent activation of Notch1 and suppressed the induction of numerous direct Notch target genes involved in lymphoid biology, lymphomagenesis and disease progression. Importantly, in lymph nodes from primary MCL cases with NOTCH1/2 mutations, we detected an upregulation of the same gene sets as observed in DLL4-stimulated Mino cells. Furthermore, DLL4 stimulation of NOTCH1-mutated Mino cells enhanced tumor cell migration and angiogenesis, which could be abolished by treatment with OMP-52M51. Importantly, the effects observed were specific for NOTCH1-mutated cells as they did not occur in the NOTCH1-wt cell line JeKo-1. Finally, we confirmed the potential activity of OMP-52M51 to inhibit DLL4-induced Notch1-Signaling in vivo in a xenograft mouse model of MCL. CONCLUSION: DLL4 effectively stimulates Notch1 signaling in NOTCH1-mutated MCL and is expressed by the microenvironment in MCL lymph nodes. Our results indicate that specific inhibition of the Notch1-ligand-receptor interaction might provide a therapeutic alternative for a subset of MCL patients.

IL-7R is essential for leukemia-initiating cell activity of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the dysregulation of signaling pathways that control intrathymic T-cell development. Relapse rates are still significant, and prognosis is particularly bleak for relapsed patients. Therefore, development of novel therapies specifically targeting pathways controlling leukemia-initiating cell (LIC) activity is mandatory for fighting refractory T-ALL. The interleukin-7 receptor (IL-7R) is a crucial T-cell developmental pathway that is commonly expressed in T-ALL and has been implicated in leukemia progression; however, the significance of IL-7R/IL-7 signaling in T-ALL pathogenesis and its contribution to disease relapse remain unknown. To directly explore whether IL-7R targeting may be therapeutically efficient against T-ALL relapse, we focused on a known Notch1-induced T-ALL model, because a majority of T-ALL patients harbor activating mutations in NOTCH1, which is a transcriptional regulator of IL-7R expression. Using loss-of-function approaches, we show that Il7r-deficient, but not wild-type, mouse hematopoietic progenitors transduced with constitutively active Notch1 failed to generate leukemia upon transplantation into immunodeficient mice, thus providing formal evidence that IL-7R function is essential for Notch1-induced T-cell leukemogenesis. Moreover, we demonstrate that IL-7R expression is an early functional biomarker of T-ALL cells with LIC potential and report that impaired IL-7R signaling hampers engraftment and progression of patient-derived T-ALL xenografts. Notably, we show that IL-7R-dependent LIC activity and leukemia progression can be extended to human B-cell acute lymphoblastic leukemia (B-ALL). These results have important therapeutic implications, highlighting the relevance that targeting normal IL-7R signaling may have in future therapeutic interventions, particularly for preventing T-ALL (and B-ALL) relapse.

MODS-Wayne, a Colorimetric Adaptation of the Microscopic-Observation Drug Susceptibility (MODS) Assay for Detection of Mycobacterium tuberculosis Pyrazinamide Resistance from Sputum Samples.

Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a color change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 µg/ml, sensitivity and specificity were evaluated at three different periods of incubation (reading 1, reading 2, and reading 3) using a composite reference standard (MGIT-PZA, pncA sequencing, and the classic Wayne test). MODS-Wayne was able to detect PZA resistance, with a sensitivity and specificity of 92.7% and 99.3%, respectively, at reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low-resource countries, where TB is a major public health problem.